We shall continue ongoing work on the characterization of new myelin enzymes, with emphasis on those involved in lipid biosynthesis. To date we have identified four enzymes, the two currently under investigation being CTP: phosphoethanolamine cytidylyltransferase and CTP: phosphocholine cytidylyltransferase. These and related enzymes are of considerable interest because of their apparent ability to utilize substrates emanating from the axon, suggesting a form of metabolic dependence of myelin on the axon. This aspect will be pursued in an effort to identify the variety of axonally-derived substrates utilized in this manner. We shall also attempt to determine the specific types of lipids synthesized in vivo by myelin-localized enzymes with substrates from the axon, and compare these to the myelin lipids synthesized by enzymes localized in the glial perikaryon. Specific new myelin enzymes will be sought based on the types of myelin lipids labeled through axonal flow of radiolabeled macromolecules. These will include choline- and ethanolamine kinase, phosphatidic acid phosphates, fatty acid CoA ligase, glycerol phosphate acyltransferase, and lysophosphatidic acid acyltransferase. Eventually we shall also look into the possible presence of certain sphingolipid synthesizing enzymes. An effort will be made to determine the localization of these enzymes within myelin by measuring enrichment in the various myelin subfractions and in myelin-related membrane fractions; comparison will be made of CNP and myelin-associated-glycoprotein, both of which are known to be enriched in periaxonal structures. Developmental profiles will be determined for a number of the myelin-localized enzymes to determine whether activity continues beyond the stage of rapid myelination. We shall carry out a detailed collaborative study on neuraminidase, recently discovered to be present in myelin, to determine its effect in generating the unique ganglioside pattern of myelin.